Hepatitis-C virus type 4, 5 and 6

ABSTRACT

The present invention relates to a polynucleic acid composition comprising or consisting of at least one polynucleic acid containing 8 or more contiguous nucleotides corresponding to a nucleotide sequence from the region spanning positions 417 to 957 of the Core/E1 region of HCV type 3; and/or the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3; and/or the region spanning positions 4892 to 5292 of the NS3/4 region of HCV type 3; and/or the region spanning positions 8023 to 8235 of the NS5 region of the BR36 subgroup of HCV type 3a; and/or the coding region of HCV type 4a starting at nucleotide 379 in the core region; and/or the coding region of HCV type 4; and/or the coding region of HCV type 5, with said nucleotide numbering being with respect to the numbering of HCV nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV type 1, and/or HCV type 2 genomes in the above-indicated regions, or the complement thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/676,842, filed Feb. 20, 2007, which is a continuation of U.S. patentapplication Ser. No. 11/090,952, filed Mar. 25, 2005, which is adivisional of U.S. patent application Ser. No. 08/537,802, filed Dec.21, 1995, which is the National Stage of International Application No.PCT/GB94/00957, filed May 5, 1994, and published in English as WO94/25602 on Nov. 10, 1994; the contents of which are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to newly elucidated sequences of hepatitisC virus type 4 (HCV-4), and type 5 (HCV-5), and to a newly discoveredtype 6 (HCV-6). In particular, it relates to the etiologic agent ofhepatitis C virus type 4, 5 and 6, and to polynucleotides andimmunoreactive polypeptides which are useful in immunoassays for thedetection of HCV-4, HCV-5 and HCV-6 in biological samples; and also tothe use of antigenic HCV-4, HCV-5 and HCV-6 specific polypeptides invaccines.

BACKGROUND OF THE INVENTION

Acute viral hepatitis is a disease which may result in chronic liverdamage. It is clinically diagnosed by a well-defined set of patientsymptoms, including jaundice, hepatic tenderness, and an increase in theserum levels of alanine aminotransferase and aspartate aminotransferase.Serologic immunoassays are generally performed to diagnose the specifictype of viral causative agent. Historically, patients presenting withsymptoms of hepatitis and not otherwise infected by hepatitis A,hepatitis B, Epstein-Barr or cytomegalovirus were clinically diagnosedas having non-A, non-B hepatitis (NANBH) by default.

For many years, the agent of non-A, non-B hepatitis remained elusive. Ithas now been established that many cases of NANBH are caused by adistinct virus termed hepatitis C virus (HCV). European PatentApplication EP-A-0318216 discloses cDNA sequences derived from onestrain of HCV, polynucleotide probes and polypeptides for use inimmunoassays. Further information on that strain is provided in EuropeanApplication EP-A-0388232.

The HCV genome encodes a large polyprotein precursor, which containsstructural and non-structural regions. The single protein is apparentlycleaved into a variety of proteins after production. Most of thestructural and non-structural proteins have now been identified from invitro RNA translation and expression as recombinant proteins. The C andE regions encode for nucleocapsid structural proteins and for envelopestructural proteins, respectively. At least five additional regionsfollow, which encode for non-structural (NS) protein of undefinedfunction. The organization is believed to be as follows (Alberti (1991)J. Hepatology 12:279-282):5′ NCR:C:E1:E2:NS1:NS2:NS3:NS4:NS5 3′

Certain immunoreactive proteins have been described as recombinantproteins, for example C22 (in the core region), C33 (in NS3 region),5-1-1 and C100 (both in the NS4 region), and NS5 (NS5 region). Currentdiagnosis of hepatitis C is often based on methods which detectantibodies against the product of the C-100 clone. This clone wasproduced by ligation of overlapping clones to produce a larger viralantigen (C100) corresponding to part of the NS3-NS4 genomic region. C100was then fused with the human superoxide dismutase (SOD) gene, expressedin use as a large recombinant fusion protein (C100-3) and used on solidphase to develop radio-labeled (RIA) and enzyme-linked immunosorbentassays (ELISA).

Polynucleotides alleged to be useful for screening for HCV are disclosedin European Patent Specification EP-A-0398748. European PatentSpecification EP-A-0414475 purports to disclose the propagation of HCVin culture cells and the production of antigens for use in diagnostics.European Patent Specification EP-A-0445423 discloses what is stated tobe an improved immunoassay for detecting HCV antibodies.

Blood banks in the United Kingdom carry out routine testing of blooddonors for antibodies to components of HCV. A first generation assayinvolved the detection of HCV antibodies to C100-3 polypeptides. TheC100-3 antibody recognizes a composite polyprotein antigen withinnon-structural regions of the virus and is a consistent marker of HCVinfection. However, in acute infections this antibody is unreliablebecause of the delay (typically 22 weeks) in seroconversion afterexposure. Furthermore, the C100-3 antibody test lacks specificity forthe hepatitis C virus.

Second generation antibody tests employ recombinant antigens and/orsynthetic linear peptides representing structural antigens from thehighly conserved core region of the virus as well as non-structuralantigens. However, it is found that some second-generation ELISA testscan yield false-positive reactions. The recombinant immunoblot assay(RIBA-2) incorporating four antigens from the HCV genome, purports toprovide a method for identifying genuine-anti-HCV reactivity. However,the result can be “indeterminate”. The present workers have reported(Chan et al. (1991) The Lancet 338:1391) varying reactivity ofHCV-positive blood donors to 5-1-1, C100, C33 and C22 antigens, andcompared these with the results of the direct detection of HCV RNApresent in the blood samples using polymerase chain reaction (PCR) toamplify HCV polynucleotides. However, the work demonstrates that theunambiguous diagnosis of HCV infections is not yet possible.

Recently there have been discovered further types of HCV that differconsiderably in sequence and these have been called HCV-2, 3 and 4. Ourpatent application WO93/10239 (published on 27 May, 1993) describescertain antigenic sequences of HCV-2, 3 and 4. The sequences disclosedfor HCV-4 are in the 5′ NCR and core regions only. The former does notcode for any protein which might be used in an immunoassay for HCVwhilst the core region tends to be conserved.

SUMMARY OF INVENTION

The present invention includes the discovery of a previously unknowntype 6 variant of HCV, by a comparison of sequences amplified bypolymerase chain reaction (PCR) in certain regions of the HCV genome andconfirmed by phylogenetic analysis. The invention has identifiedpolynucleotide sequences and polypeptides which are HCV-4, HCV-5 andHCV-6 specific. These may be used to diagnose HCV-4, HCV-5 and HCV-6infection and should thus be included in any definitive test for HCVinfection.

One aspect of the present invention provides a polynucleotide having anucleotide sequence unique to hepatitis virus type 4, 5 or 6. Thesequences are unique to the HCV type concerned in the sense that thesequence is not shared by any other HCV type, and can thus be used touniquely detect that HCV-type. Sequence variability between HCV 4, 5 and6 has been found particularly in the NS4, NS5 and core regions and it istherefore from these regions in particular that type-specificpolynucleotides and peptides may be obtained. The term type-specificindicates that a sequence unique to that HCV type is involved. Moreover,within each HCV type a number of sub-types may exist having minorsequence variations.

The invention includes NS5 polynucleotide sequences unique to hepatitisC virus types 4 and 6 (HCV-4 and HCV-6); and NS4 sequences unique toHCV-4, HCV-5 and HCV-6 respectively. The sequences may be RNA or DNAsequences, including cDNA sequences. If necessary the DNA sequences maybe amplified by polymerase chain reaction. DNA sequences can be used asa hybridization probe. The sequences may be recombinant (i.e. expressedin transformed cells) or synthetic and may be comprised within longersequences if necessary. Equally, deletions, insertions or substitutionsmay also be tolerated if the polynucleotide may still function as aspecific probe. Polynucleotide sequences which code for antigenicproteins are also particularly useful.

Another aspect of the invention provides a peptide having an amino acidsequence unique to hepatitis virus type 4, 5 or 6.

The invention includes antigenic HCV-4 or HCV-6 specific polypeptidefrom the NS5 region, or antigenic HCV-4, HCV-5 or HCV-6 specificpolypeptide from the NS4 region; or polypeptides including theseantigens. A plurality of copies of the peptide may be bound to amultiple antigen peptide core.

The peptide may be labeled to facilitate detection, and may for examplebe labeled antigenic HCV-4 or HCV-6 specific polypeptide from the NS5region, or labeled antigenic HCV-4, HCV-5 or HCV-6 specific polypeptidefrom the NS4 region; (or mixtures thereof) for use in an immunoassay todetect the corresponding antibodies.

It should be understood that the polypeptides will not necessarilycomprise the entire NS4 or NS5 region, but that characteristic partsthereof (usually characteristic epitopes) unique to a particular type ofHCV may also be employed.

A further aspect of the invention provides antibodies to the peptides,especially to HCV-4 or HCV-6 NS5 antigens, or to HCV-4, HCV-5 or HCV-6NS4 antigens, particularly monoclonal antibodies for use in therapy anddiagnosis. Thus labeled antibodies may be used for in vivo diagnosis.Antibodies carrying cytotoxic agents may be used to attack HCV-4, HCV-5or HCV-6 infected cells.

A further aspect of the invention provides a vaccine comprisingimmunogenic peptide, especially HCV-4 or HCV-6 NS5 polypeptide, orimmunogenic HCV-4, HCV-5 or HCV-6 NS4 polypeptide.

A further aspect of the invention provides a method of in vitro HCVtyping which comprises carrying out endonuclease digestion of anHCV-containing sample to provide restriction fragments, the restrictionpattern being characteristic of HCV-4, HCV-5 or HCV-6.

Finally, the present invention also encompasses assay kits includingpolypeptides which contain at least one epitope of HCV-4, HCV-5 or HCV-6antigen (or antibodies thereto), as well as necessary preparativereagents, washing reagents, detection reagents and signal producingreagents.

DESCRIPTION OF FIGURES

FIG. 1 is a comparison of inferred amino acid sequences of part of theNS5 region of HCV types 4 (SEQ ID NOS:14 and 16) and 6 (SEQ ID NO:18)(in comparison to type la (SEQ ID NOS:1-9)) from Example 1. The numberof sequences compared is shown in the second column. Single amino acidcodes are used. The position and frequency of variability within an HCVtype is indicated by a subscript.

FIG. 2 gives corresponding DNA nucleotide sequences in the NS5 region ofHCV-4 (SEQ ID NOS:10, 13, and 15) and of HCV-6 (SEQ ID NO:17).

FIG. 3 is a phylogenetic analysis of NS5 sequences from 67 isolates ofHCV, showing major HCV types (numbered 1-6) and subtypes (designateda,b,c) and demonstrating that HCV-4 and HCV-6 are distinct types. Thesequence distance is proportional to the spacing on the tree accordingto the indicated scale.

FIG. 4 shows DNA primer sequences (SEQ ID NOS:19-22) as used in Example2 for the PCR amplification of two regions of the NS4 region of HCV-4,HCV-5 and HCV-6.

FIG. 5 shows DNA and amino acid sequences for two partial regions of theNS4 region of HCV-4 (NS4 Region 1: SEQ ID NOS:31 and 32; NS4 Region 2:SEQ ID NOS:45 and 46), HCV-5 (NS4 Region 1: SEQ ID NOS:33 and 34; NS4Region 2: SEQ ID NOS:47 and 48) and HCV-6 (NS4 Region 1: SEQ ID NOS:35and 36; NS4 Region 2: SEQ ID NOS:49 and 50) deduced from the nucleotidesequences elucidated in Example 2; for comparison the correspondingregions of HCV-1 (NS4 Region 1: SEQ ID NOS:23, 24, 25 and 26; NS4 Region2: SEQ ID NOS:37, 38, 39 and 40), HCV-2 (NS4 Region 1: SEQ ID NOS:27 and28; NS4 Region 2: SEQ ID NOS:41 and 42) and HCV-3 (NS4 Region 1: SEQ IDNOS:29 and 30; NS4 Region 2: SEQ ID NOS:43 and 44) are given, HCV-3regions 1 and 2 corresponding to amino acids 1691-1708 and 1710-1728respectively of FIG. 9 b of W093/10239 (see also Simmonds et al. (1993)J. Clin. Microb. 31:1493).

DETAILED DESCRIPTION

The HCV-4, HCV-5 or HCV-6 specific polynucleotide sequences may be usedfor identification of the HCV virus itself (usually amplified by PCR) byhybridization techniques.

Oligonucleotides corresponding to variable regions, e.g. in the NS5 orNS4 region, could be used for type-specific PCR. Outer sense and innersense primers may be used in combination with the two conservedanti-sense primers for a specific detection method for HCV types 4, 5and 6.

The present invention also provides expression vectors containing theDNA sequences as herein defined, which vectors being capable, in anappropriate host, of expressing the DNA sequence to produce the peptidesas defined herein. The expression vector normally contains controlelements of DNA that effect expression of the DNA sequence in anappropriate host. These elements may vary according to the host butusually include a promoter, ribosome binding site, translational startand stop sites, and a transcriptional termination site. Examples of suchvectors include plasmids and viruses. Expression vectors of the presentinvention encompass both extrachromosomal vectors and vectors that areintegrated into the host cell's chromosome. For use in E. coli, theexpression vector may contain the DNA sequence of the present inventionoptionally as a fusion linked to either the 5′- or 3′-end of the DNAsequence encoding, for example, B-galactosidase or the 3′-end of the DNAsequence encoding, for example, the trp E gene.

For use in the insect baculovirus (AcNPV) system, the DNA sequence isoptionally fused to the polyhedrin coding sequence.

The present invention also provides a host cell transformed withexpression vectors as herein defined. Examples of host cells of use withthe present invention include prokaryotic and eukaryotic cells, such asbacterial, yeast, mammalian and insect cells. Particular examples ofsuch cells are E. coli, S. cerevisiae, P. pastoris, Chinese hamsterovary and mouse cells, and Spodoptera frugiperda and Tricoplusia ni. Thechoice of host cell may depend on a number of factors but, ifpost-translational modification of the HCV viral peptide is important,then a eukaryotic host would be preferred.

The present invention also provides a process for preparing a peptide asdefined herein which comprises isolating the DNA sequence, as hereindefined, from the HCV genome, or synthesizing DNA sequence encoding thepeptides as defined herein, or generating a DNA sequence encoding thepeptide, inserting the DNA sequence into an expression vector such thatit is capable, in an appropriate host, of being expressed, transforminghost cells with the expression vector, culturing the transformed hostcells, and isolating the peptide.

The DNA sequence encoding the peptide may be synthesized using standardprocedures (Gait, Oligonucleotide Synthesis: A Practical Approach, 1984,Oxford, IRL Press).

The desired DNA sequence obtained as described above may be insertedinto an expression vector using known and standard techniques. Theexpression vector is normally cut using restriction enzymes and the DNAsequence inserted using blunt-end or staggered-end ligation. The cut isusually made at a restriction site in a convenient position in theexpression vector such that, once inserted, the DNA sequences are underthe control of the functional elements of DNA that effect itsexpression.

Transformation of a host cell may be carried out using standardtechniques. Some phenotypic marker is usually employed to distinguishbetween the transformants that have successfully taken up the expressionvector and those that have not. Culturing of the transformed host celland isolation of the peptide as required may also be carried out usingstandard techniques.

The peptides of the present invention may thus be prepared byrecombinant DNA technology, or may be synthesized, for example by usingan automatic synthesizer. The term “peptide” (and “polypeptide”) is usedherein to include epitopic peptides having the minimum number of aminoacid residues for antigenicity, through oligopeptides, up to proteins.The peptide may be a recombinant peptide expressed from a transformedcell, or could be a synthetic peptide produced by chemical synthesis.

Antibody specific to a peptide of the present invention can be raisedusing the peptide. The antibody may be used in quality control testingof batches of the peptides; purification of a peptide or viral lysate;epitope mapping; when labeled, as a conjugate in a competitive typeassay, for antibody detection; and in antigen detection assays.

Polyclonal antibody against a peptide of the present invention may beobtained by injecting a peptide, optionally coupled to a carrier topromote an immune response, into a mammalian host, such as a mouse, rat,sheep or rabbit, and recovering the antibody thus produced. The peptideis generally administered in the form of an injectable formulation inwhich the peptide is admixed with a physiologically acceptable diluent.Adjuvants, such as Freund's complete adjuvant (FCA) or Freund'sincomplete adjuvant (FIA), may be included in the formulation. Theformulation is normally injected into the host over a suitable period oftime, plasma samples being taken at appropriate intervals for assay foranti-HCV viral antibody. When an appropriate level of activity isobtained, the host is bled. Antibody is then extracted and purified fromthe blood plasma using standard procedures, for example, by protein A orion-exchange chromatography.

Monoclonal antibody against a peptide of the present invention may beobtained by fusing cells of an immortalizing cell line with cells whichproduce antibody against the viral of topographically related peptide,and culturing the fused immortalized cell line. Typically, a non-humanmammalian host, such as a mouse or rat, is inoculated with the peptide.After sufficient time has elapsed for the host to mount an antibodyresponse, antibody producing cells, such as the splenocytes, areremoved. Cells of an immortalizing cell line, such as a mouse or ratmyeloma cell line, are fused with the antibody producing cells and theresulting fusions screened to identify a cell line, such as a hybridoma,that secretes the desired monoclonal antibody. The fused cell line maybe cultured and the monoclonal antibody purified from the culture mediain a similar manner to the purification of polyclonal antibody.

Diagnostic assays based upon the present invention may be used todetermine the presence of absence of HCV infection, and the HCV typeinvolved. They may also be used to monitor treatment of such infection,for example in interferon therapy. In an assay for the diagnosis ofviral infection, there are basically three distinct approaches that canbe adopted involving the detection of viral nucleic acid, viral antigenor viral antibody respectively. Viral nucleic acid is generally regardedas the best indicator of the presence of the virus itself and wouldidentify materials likely to be infectious. However, the detection ofnucleic acid is not usually as straightforward as the detection ofantigens or antibodies since the level of target can be very low. Viralantigen is used as a marker for the presence of virus and as anindicator of infectivity. Depending upon the virus, the amount ofantigen present in a sample can be very low and difficult to detect.Antibody detection is relatively straightforward because, in effect, thehost immune system is amplifying the response to an infection byproducing large amounts of circulating antibody. The nature of theantibody response can often be clinically useful, for example IgM ratherthan IgG class antibodies are indicative of a recent infection, or theresponse to a particular viral antigen may be associated with clearanceof the virus. Thus the exact approach adopted for the diagnosis of aviral infection depends upon the particular circumstances and theinformation sought. In the case of HCV, a diagnostic assay may embodyany one of these three approaches.

In any assay for the diagnosis of HCV involving detection of viralnucleic acid, the method may comprise hybridizing viral RNA present in atest sample, or cDNA synthesized from such viral RNA, with a DNAsequence corresponding to the nucleotide sequences of the presentinvention or encoding a peptide of the invention, and screening theresulting nucleic acid hybrids to identify any HCV viral nucleic acid.The application of this method is usually restricted to a test sample ofan appropriate tissue, such as a liver biopsy, in which the viral RNA islikely to be present at a high level. The DNA sequence corresponding toa nucleotide sequence of the present invention or encoding a peptide ofthe invention may take the form of an oligonucleotide or a cDNA sequenceoptionally contained within a plasmid. Screening of the nucleic acidhybrids is preferably carried out by using a labeled DNA sequence.Preferably the peptide of the present invention is part of anoligonucleotide wherein the label is situated at a sufficient distancefrom the peptide so that binding of the peptide to the viral nucleicacid is not interfered with by virtue of the label being too close tothe binding site. One or more additional rounds of screening of one kindor another may be carried out to characterize further the hybrids andthus identify any HCV viral nucleic acid. The steps of hybridization andscreening are carried out in accordance with procedures known in theart.

A further method for the detection of viral nucleic acid involvesamplification of a viral DNA using polymerase chain reaction (PCR). Theprimers chosen may be specific to the HCV type sequence of interest, sothat amplification occurs only with that particular HCV type. Also thesize and number of amplified copy sequences may be characteristic ofparticular HCV types, or they may have characteristic restrictionpatterns with chosen endonucleases.

In an assay for the diagnosis of HCV involving detection of viralantigen or antibody, the method may comprise contacting a test samplewith a peptide of the present invention or a polyclonal or monoclonalantibody against the peptide and determining whether there is anyantigen-antibody binding contained within the test sample. For thispurpose, a test kit may be provided comprising a peptide, as definedherein, or a polyclonal or monoclonal antibody thereto and means fordetermining whether there is any binding with antibody or antigenrespectively contained in the test sample to produce an immune complex.The test sample may be taken from any of appropriate tissue orphysiological fluid, such as blood (serum or plasma), saliva, urine,cerebrospinal fluid, sweat, tears or tissue exudate. If a physiologicalfluid is obtained, it may optionally be concentrated for any viralantigen or antibody present.

A variety of assay formats may be employed. The peptide can be used tocapture selectively antibody against HCV from solution, to labelselectively the antibody already captured, or both to capture and labelthe antibody. In addition, the peptide may be used in a variety ofhomogeneous assay formats in which the antibody reactive with thepeptide is detected in solution with no separation of phases.

The types of assay in which the peptide is used to capture antibody fromsolution involve immobilization of the peptide on to a solid surface.This surface should be capable of being washed in some way. Examples ofsuitable surfaces include polymers of various types (molded intomicrotiter wells; beads; dipsticks of various types; aspiration tips;electrodes; and optical devices); particles (for example latex;stabilized red blood cells; bacterial or fungal cells; spores; gold orother metallic or metal-containing sols; and proteinaceous colloids)with the usual size of the particle being from 0.02 to 5 microns;membranes (for example of nitrocellulose; paper; cellulose acetate; andhigh porosity/high surface area membranes of an organic or inorganicmaterial).

The attachment of the peptide to the surface can be by passiveadsorption from a solution of optimum composition which may includesurfactants, solvents, salts and/or chaotropes; or by active chemicalbonding. Active bonding may be through a variety of reactive oractivatable functional groups which may be exposed on the surface (forexample condensing agents; active acid esters, halides and anhydrides;amino, hydroxyl, or carboxyl groups; sulphydryl groups; carbonyl groups;diazo groups; or unsaturated groups). Optionally, the active bonding maybe through a protein (itself attached to the surface passively orthrough active bonding), such as albumin or casein, to which the viralpeptide may be chemically bonded by any of a variety of methods. The useof a protein in this way may confer advantages because of isoelectricpoint, charge, hydrophilicity or other physico-chemical property. Theviral peptide may also be attached to the surface (usually but notnecessarily a membrane) following electrophoretic separation of areaction mixture, such as immunoprecipitation.

In the present invention it is preferred to provide blocking peptideswhich block any cross-reactivity and leave only those HCV antibodies inthe sample which will react solely with the type of antigen present inthat particular test location. For example, a test location intended todetect HCV-6 will be blocked by a blocking mixture comprising HCV-1 to 5peptides which will react with all antibodies having reactivity to HCVtypes 1 to 5 and leave antibodies having only type 6 reactivity.

After contacting the surface bearing the peptide with a test sample (inthe presence of a blocking mixture if required), allowing time forreaction, and, where necessary, removing the excess of the sample by anyof a variety of means, (such as washing, centrifugation, filtration,magnetism or capillary action) the captured antibody is detected by anymeans which will give a detectable signal. For example, this may beachieved by use of a labeled molecule or particle as described abovewhich will react with the captured antibody (for example protein A orprotein G and the like; anti-species or anti-immunoglobulin-sub-type;rheumatoid factor; or antibody to the peptide, used in a competitive orblocking fashion), or any molecule containing an epitope contained inthe peptide. In the present invention, it is preferred to add ananti-human IgG conjugated to horseradish peroxidase and then to detectthe bound enzyme by reaction with a substrate to generate a color.

The detectable signal may be produced by any means known in the art suchas optical or radioactive or physico-chemical and may be provideddirectly by labeling the molecule or particle with, for example, a dye,radiolabel, fluorescent, luminescent, chemiluminescent, electroactivespecies, magnetically resonant species or fluorophore, or indirectly bylabeling the molecule or particle with an enzyme itself capable ofgiving rise to a measurable change of any sort. Alternatively thedetectable signal may be obtained using, for example, agglutination, orthrough a diffraction or birefrigent effect if the surface is in theform of particles.

Assays in which a peptide itself is used to label an already capturedantibody require some form of labeling of the peptide which will allowit to be detected. The labeling may be direct by chemically or passivelyattaching for example a radiolabel, magnetic resonant species, particleof enzyme label to the peptide; or indirect by attaching any form oflabel to a molecule which will itself react with the peptide. Thechemistry of bonding a label to the peptide can be directly through amoiety already present in the peptide, such as an amino group, orthrough an intermediate moiety, such as a maleimide group. Capture ofthe antibody may be on any of the surfaces already mentioned in anyreagent including passive or activated adsorption which will result inspecific antibody or immune complexes being bound. In particular,capture of the antibody could be by anti-species oranti-immunoglobulin-sub-type, by rheumatoid factor, proteins A, G andthe like, or by any molecule containing an epitope contained in thepeptide.

The labeled peptide may be used in a competitive binding fashion inwhich its binding to any specific molecule on any of the surfacesexemplified above is blocked by antigen in the sample. Alternatively, itmay be used in a non-competitive fashion in which antigen in the sampleis bound specifically or non-specifically to any of the surfaces aboveand is also bound to a specific bi- or poly-valent molecule (e.g. anantibody) with the remaining valencies being used to capture the labeledpeptide.

Often in homogeneous assays the peptide and an antibody are separatelylabeled so that, when the antibody reacts with the recombinant peptidein free solution, the two labels interact to allow, for example,non-radiative transfer of energy captured by one label to the otherlabel with appropriate detection of the excited second label or quenchedfirst label (e.g. by fluorimetry, magnetic resonance or enzymemeasurement). Addition of either viral peptide or antibody in a sampleresults in restriction of the interaction of the labeled pair and thusin a different level of signal in the detector.

A further possible assay format for detecting HCV antibody is the directsandwich enzyme immunoassay (EIA) format. An antigenic peptide is coatedonto microtiter wells. A test sample and a peptide to which an enzyme iscoupled are added simultaneously. Any HCV antibody present in the testsample binds both to the peptide coating the well and to theenzyme-coupled peptide. Typically, the same peptide are used on bothsides of the sandwich. After washing, bound enzyme is detected using aspecific substrate involving a color change.

It is also possible to use IgG/IgM antibody capture ELISA wherein anantihuman IgG and/or IgM antibody is coated onto a solid substrate. Whena test sample is added, IgG and/or IgM present in the sample will thenbind to the antihuman antibody. The bound IgG and/or IgM represents thetotal population of those antibodies. A peptide of the present inventionwill bind only to those IgG and/or IgM antibodies that were produced inresponse to the antigenic determinant(s) present in the peptide i.e. tothose antibodies produced as a result of infection with the type of HCVfrom which the peptide was derived. For detection of thepeptide/antibody complex the peptide may itself have been labeleddirectly or, after interaction with the captures antibodies, the peptidemad be reacted with a labeled molecule that binds to the peptide.

It can thus be seen that the peptides of the present invention may beused for the detection of HCV infection in many formats, namely as freepeptides, in assays including classic ELISA, competition ELISA, membranebound EIA and immunoprecipitation. Peptide conjugates may be used inamplified assays and IgG/IgM antibody capture ELISA.

An assay of the present invention may be used, for example, forscreening donated blood or for clinical purposes, for example, in thedetection, typing and monitoring of HCV infections. For screeningpurposes, the preferred assay formats are those that can be automated,in particular, the microtiter plate format and the bead format. Forclinical purposes, in addition to such formats, those suitable forsmaller-scale or for single use, for example, latex assays, may also beused. For confirmatory assays in screening procedures, antigens may bepresented on a strip suitable for use in Western or other immunoblottingtests.

As indicated above, assays used currently to detect the presence ofanti-HCV antibodies in test samples, particularly in screening donatedblood, utilize antigenic peptides obtained from HIV type 1 only and suchantigens do not reliable detect other HCV genotypes. Accordingly, it isclearly desirable to supplement testing for HIV-1 with testing for allother genotypes, for example, types 2, 3, 4, 5 and 6 and also anyfurther genotypes that may be discovered.

In particular, the invention allows blood donor screening byconventional assays (using HCV type 1 encoded antigens) to besupplemented with a second test that contains oligopeptidescorresponding to antigenic regions found for example in the NS5 sequenceof HCV-4 or HCV-6, or the NS4 sequence of HCV-4, HCV-5 or HCV-6.

To test for a spectrum of genotypes, there may be provided a series ofassay means each comprising one or more antigenic peptides from onegenotype of HCV, for example, a series of wells in a microtiter plate,or an equivalent series using the bead format. Such an assay format maybe used to determine the type of HCV present in a sample. Alternatively,or in addition, an assay means may comprise antigenic peptides from morethan one type, for example, a microwell or bead may be coated withpeptides from more than one type.

Oligopeptides corresponding to the antigenic regions of HCV-4, HCV-5 orHCV-6 may also be used separately to distinguish individuals infectedwith these different HCV types. Such an assay could be in the format ofan indirect enzyme immunoassay (EIA) that used sets of wells or beadscoated with oligopeptides of the antigenic regions for HCV types 4, 5and 6. Minor degrees of cross-reactivity, should they exist, can beabsorbed out by dilution of the test serum in a diluent that containedblocking amounts of soluble heterologous-type oligopeptides, to ensurethat only antibody with type-specific antibody reactivity bound to thesolid phase.

It may be advantageous to use more than one HCV antigen for testing, inparticular, a combination comprising at least one antigenic peptidederived from the structural region of the genome and at least oneantigenic peptide derived from the non-structural region, especially acombination of a core antigen and at least one antigen selected from theNS3, NS4 and NS5 regions. The wells or beads may be coated with theantigens individually. It may be advantageous, however, to fuse two ormore antigenic peptides as a single polypeptide, preferably as arecombinant fusion polypeptide. Advantages of such an approach are thatthe individual antigens can be combined in a fixed, predetermined ratio(usually equimolar) and that only a single polypeptide needs to beproduced, purified and characterized. One or more such fusionpolypeptides may be used in an assay, if desired in addition to one ormore unfused peptides. It will be appreciated that there are manypossible combinations of antigens in a fusion polypeptide, for example,a fusion polypeptide may comprise a desired range of antigens from onetype only, or may comprise antigens from more than one type.

To obtain a polypeptide comprising multiple peptide antigens by anexpression technique, one approach is to fuse the individual codingsequences into a single open reading frame. The fusion should, ofcourse, be carried out in such a manner that the antigenic activity ofeach component peptide is not significantly compromised by its positionrelative to another peptide. Particular regard should of course be hadfor the nature of the sequences at the actual junction between thepeptides. The resulting coding sequence can be expressed, for example,as described above in relation to recombinant peptides in general. Themethods by which such a fusion polypeptide can be obtained are known inthe art, and the production of a recombinant fusion polypeptidecomprising multiple antigens of a strain of HCV type 1 is described inGB-A-2 239 245. Peptide conjugates may be used in amplified assays andIgG/IgM antibody capture ELISA.

The peptide of the present invention may be incorporated into a vaccineformulation for inducing immunity to HCV in man. The vaccine may includeantigens of HCV types 1 to 6. For this purpose the peptide may bepresented in association with a pharmaceutically acceptable carrier. Foruse in a vaccine formulation, the peptide may optionally be presented aspart of a hepatitis B core fusion particle, as described in Clarke etal. (1987) Nature 330:381-384, or a polylysine based polymer, asdescribed in Tam (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413.Alternatively, the peptide may optionally be attached to a particulatestructure, such as lipsomes or ISCOMS.

Pharmaceutically, acceptable carriers for the vaccine include liquidmedia suitable for use as vehicles to introduce the peptide into apatient. An example of such liquid media is saline solution. The peptidemay be dissolved or suspended as a solid in the carrier. The vaccineformulation may also contain an adjuvant for stimulating the immuneresponse and thereby enhancing the effect of the vaccine. Examples ofadjuvants include aluminum hydroxide and aluminum phosphate. The vaccineformulation may contain a final concentration of peptide in the rangefrom 0.01 to 5 mg/ml, preferably from 0.03 to 2 mg/ml. The vaccineformulation may be incorporated into a sterile container, which is thensealed and stored at a low temperature, for example 4 degree C., or maybe freeze-dried.

In order to induce immunity in man to HCV, one or more doses of thevaccine formulation may be administered. Each dose may be 0.1 to 2 ml,preferably 0.2 to 1 ml. A method for inducing immunity to HCV in man,comprises the administration of an effective amount of a vaccineformulation, as hereinbefore defined. The present invention alsoprovides the use of a peptide as herein defined in the preparation of avaccine for use in the induction of immunity to HCV in man. Vaccines ofthe present invention may be administered by any convenient method forthe administration of vaccines including oral and parenteral (e.g.intravenous, subcutaneous or intramuscular) injection. The treatment mayconsist of a single dose of vaccine or a plurality of doses over aperiod of time.

Examples of the invention will now be described by way of example only.

EXAMPLE 1 HCV-4 and HCV-6; NS5 Region Sequences

Samples. Plasma from a total of 16 HCV-infected blood donors inScotland, Egypt and Hong Kong and from a patient with chronic hepatitisin Lebabon were used for analysis of NS5 sequences of types 4 and 6.

Nucleotide sequence analysis. To obtain sequences in the NS5 region,viral RNA was reverse transcribed and amplified by polymerase chainreaction (PCR) in a single reaction with previously published primersthought to be highly conserved amongst different variants of HCV(Enomoto et al. (1990) Biochemical and Biophysical ResearchCommunications 170:1021-1025). For some sequences, a second PCR wascarried out with primers 554 and 555 (Chan et al. (1992)J. Gen. Virol.73:1131-1141) in combination with two new primers, 122 (senseorientation; 5′ CTC AAC CGT CAC TGA GAG AGA CAT 3′) (SEQ ID NO:51) and123 (anti-sense; 5′ GCT CTC AGG TTC CGC TCG TCC TCC 3′) (SEQ ID NO:52).Product DNA was phosphorylated, purified and cloned into the SmaI siteof pUC19 (Yanisch-Perron et al. (1985) Gene 33:103-107). following theprocedures described in Simmonds & Chan (1993) Analysis of viralsequence variation by PCR. In Molecular Virology: A Practical Approach,pp. 109-138. Edited by A. J. Davidson and R. M. Elliot. Oxford IRLPress. Alternatively, amplified DNA was purified and directly sequencedas described in Simmonds et al. (1990) J. Virol. 64:5840-5850 and Cha etal. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7144-7148. These methodsallowed comparison of a 222 bp fragment of DNA homologous to positions7975 to 8196 in the prototype virus (numbered as in Choo et al. (1991)Proc. Natl. Acad. Sci. U.S.A. 88:2451-2455). The results are shown inFIGS. 1 and 2.

EXAMPLE 2 HCV-4, -5 and -6; NS4 Region Sequences

Attempts have been made to isolate DNA sequences from the NS4 region ofHepatitis C virus (HCV) types 4, 5 and 6 using PCR amplification. Thedecision was made to use primers which contained restriction sites,thereby allowing the cloning of the PCR products via cohesive endcloning. New primers were also designed from relatively conservedregions of the HCV NS4 gene. The cloning strategy involved severalparticular steps, as follows.

(i) Klenow repair. The termini of the amplified DNA were repaired withKlenow DNA polymerase to ensure that the ends which contained therestriction sites were complete.

(ii) Kinasing of termini. The PCR product termini were phosphorylatedusing T4 polynucleotide kinase. This allowed self-ligation of theproducts in a concatemerization step.

(iii) Concatemerization of the PCR products. The DNA fragments wereligated together to form long concatemeric arrays. This stepinternalized the restriction sites encoded in the ends of the primerswhich greatly increased the efficiency of the cleavage step.

(iv) Restriction digestion. The PCR products were digested overnightwith the required restriction enzyme to expose the cohesive ends.

(v) Ligation to the plasmid vector.

General procedures and reagents are described in Maniatis et al.“Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory,Cold Spring Harbor: N.Y.

PCR Amplification of NS4 Sequences

After cDNA synthesis using primer 007, three rounds of PCR amplificationwere carried out. Types 4 and 5 were amplified by one round using 007,and 435, followed by two rounds using 5351 and 5943 (which both encodeBamHI restriction sites). Type 6 sequences were amplified by one roundusing 007 plus 253, 281 and 221, followed by two rounds using 5351 and5943. The products of a minimum of 5 third round reactions were pooledfor the cloning steps. The efficiency of the final ligation into thevector appeared to be dependant on a high concentration of the amplifiedDNA. The primer sequences are listed in FIG. 4.

Cloning

The PCR products were isolated by excision from a conventional agarosegel (0.5× tris acetate EDTA (TAE)). The DNA was reclaimed from theagarose by centrifugation through glass wool. The eluates were pooled inorder to increase the total amount of DNA. The DNA was retrieved fromthe TAE eluate by ethanol precipitation. (The pelleted DNA containedsome chemically inert debris from the agarose but this did not interferewith the following steps prior to ligation into the vector. Magic prepcolumns (Promega) could be used, but ethanol precipitation is simpler,cheaper and more efficient if dealing with a large volume of TAEeluate).

Klenow Repair

The precipitated DNA pellets were resuspended in a 50 ul Klenow reactionmixture comprising:

-   -   5 ul polynucleotide kinase buffer (10×),    -   0.5 ul 3.3 mM dNTPs (final concentration 33 uM),    -   at least 100 ng of purified PCR fragment,    -   distilled water to 50 ul, and    -   5 units Klenow DNA polymerase        (10× means that the reagent was added at 10 times the desired        final concentration in the reaction mixture)

Incubation was carried out for 30 min at 37 degree C., followed by heatinactivation for 10 min at 75 degree C. The Klenow reaction repairs theends where the BamHI restriction sites are located. T4 DNA polymeraseshould not be used for this reaction as it will remove the sites by itsexonuclease activity.

Kinasing of Ends

To the above reaction mixture, the following were added:

-   -   5 ul 100 mM rATP, and    -   10 units T4 polynucleotide kinase        This reaction mixture was incubated at 37 degree C. for 30-60        min and heat inactivated as before.        Concatemerization

The PCR products were then concatemerized to internalize the BamHIrestriction sites within large multimers. To the phosphorylationreaction, the following were added:

-   -   6 ul 10× ligase buffer, and    -   5 units of T4 DNA ligase        The ligation was incubated overnight at 15 degree C. The        concatemerization reaction was heat inactivated as described        previously.        Restriction Digestion

The concatemerized PCR products were digested into monomers using BamHIrestriction enzyme which simultaneously exposed the cohesive termini.The following were added to the heat inactivated ligation reactionmixture:

-   -   6 ul 10× B buffer (Boehringer), and    -   10-20 units BamHI        The digestion mixture was incubated overnight at 37 degree C.        Although the enzyme is not totally inactivated by heat, the        reaction mixture was heat treated as before anyway. At this        point, the DNA was purified by a Magic prep column to remove        cleaved ends. The DNA was eluted from the Magic prep column in        10 ul in order to be as concentrated as possible.        Ligation to Vector

100 ng of bacterial plasmid vector pUC18 was used in the ligation. Theplasmid vector DNA had been BamHI-cleaved and purified using “Geneclean”(Bio 101), but not dephosphorylated. (We found that thedephosphorylation reaction lowered the ligation efficiency of the vectorto a great extent.) It was planned that blue-white color selection asdescribed hereafter would be sufficient to identify clones. The ligationreaction mixture contained the following:

-   -   110 ul of purified insert DNA produced as above    -   5 ul plasmid vector DNA,    -   1.5 ul 10× ligase buffer, and    -   1 unit of ligase        The reaction mixture was incubated overnight at 15 degree C.        Transformation of E. coli

Bacterial transformations were carried out using the cell strain, XL-1Blue (Stratagene). Cells were made competent for transformation bystandard calcium chloride methods and stored quick-frozen in aglycerol/calcium chloride suspension in 200 ul aliquots. 3 ul of theligation reaction product were used to transform 100 ul of rapidlythawed competent XL-1 Blue cells (10 min on ice, 2 min at 42 degree C.,1 hour at 37 degree C. with shaking after addition of 1 ml of L broth).The cells were plated in 200 ul aliquots onto L agar plates containingX-Gal (20 ug/ml), IPTG (0.1 mM), Ap (50 ug/ml) and Tet (12.5 ug/ml). Thepresence of the chemicals IPTG(isopropyl-.beta.-D-thiogalactopyranoside) and X-gal(5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside) in the mediaproduces a blue color in colonies which contain plasmids such as the pUCseries which encode the lacZ enzymic peptide. The insertion of clonedDNA into the polylinker region of these plasmids interrupts the lacZpeptide sequence, thereby destroying the ability of the plasmid toproduce the enzyme. Bacterial colonies which contain recombinantplasmids will, therefore, be white.

Analysis of White Colonies

Blue-white selection identified cell colonies which containedrecombinant plasmids. These colonies were picked and DNA was preparedfrom them by mini-plasmid preparations. Digestion of the DNA with BamHIconfirmed that the plasmids contained cloned inserts. The plasmid DNAwas purified by glassmilk and sequenced using the USB Sequenase kit withM13 forward and reverse primers.

Discussion

Although this protocol contains a large number of steps, it is simple toexecute. Further work has found that the method has a high degree ofreproducibility. Theoretically, this cloning strategy should not work ifthe PCR product contains an internal BamHI site. However, the NS4sequence which was obtained for type 6 did contain such an internalsite; and there are no obvious reasons why foreshortened type 6sequences were not in fact the predominant product of the cloningexperiment.

FIG. 5 shows the DNA and amino acid sequences for two regions or NS4 inrespect at HCV types 1 to 6 (for comparison) and types 4 to 6.

EXAMPLE 3 Synthesis of NS4 Peptides

The following peptides within the NS4 region of HCV types 1 to 6 weresynthesized. Types 4 to 6 are novel sequences according to the presentinvention, whereas types 1 to 3 are presented for comparison purposesand use in a complete HCV serotyping assay.

HCV type 1 (SEQ ID NO:26) [H₂N-KPAIIPDREVLYREFDEM]₈K₄K₂K-COOH (MDL031)HCV type 1 (SEQ ID NO:24) [H₂N-KPAVIPDREVLYREFDEM]₈K₄K₂K-COOH (MDL033)HCV type 1 (SEQ ID NO:53) [H₂N-RPAVIPDREVLYQEFDEM]₈K₄K₂K-COOH (MDL036)HCV type 1 (SEQ ID NO:54) [H₂N-RPAVVPDREVLYQEFDEM]₈K₄K₂K-COOH (MDL035)HCV type 1 (SEQ ID NO:55) [H₂N-ECSQHLPYIEQGMMLAEQF]₈K₄K₂K-COOH (MDL037Q)HCV type 1 (SEQ ID NO:56) [H₂N-ECSQHLPYIEQGMALAEQF]₈K₄K₂K-COOH (MDL038Q)HCV type 2 (SEQ ID NO:57) [H₂N-RVVVTPDKEILYEAFDEM]₈K₄K₂K-COOH (MDL039)HCV type 2 (SEQ ID NO:42) [H₂N-ECASRAALIEEGQRIAEML]₈K₄K₂K-COOH (MDL041)HCV type 2 (SEQ ID NO:58) [H₂N-ECASKAALIEEGQRMAEML]₈K₄K₂K-COOH (MDL040)HCV type 3 (SEQ ID NO:30) [H₂N-KPALVPDKEVLYQQYDEM]₈K₄K₂K-COOH (MDL042)HCV type 3 (SEQ ID NO:44) [H₂N-ECSQAAPYIEQAQVIAHQF]₈K₄K₂K-COOH (MDL044)HCV type 4 (SEQ ID NO:32) [H₂N-QPAVIPDREVLYQQFEDEM]₈K₄K₂K-COOH (MDL034)HCV type 4 (SEQ ID NO:46) [H₂N-ECSKHLPLVEHGLQLAEQF]₈K₄K₂K-COOH (MDL028)HCV type 5 (SEQ ID NO:34) [H₂N-RPAIIPDREVLYQQFDKM]₈K₄K₂K-COOH (MDL024)HCV type 5 (SEQ ID NO:48) [H₂N-ECSTSLPYMDEARAIAGQF]₈K₄K₂K-COOH (MDL029)HCV type 6 (SEQ ID NO:36) [H₂N-KPAVVPDREILYQQFDEM]₈K₄K₂K-COOH (MDL025)HCV type 6 (SEQ ID NO:50) [H₂N-ECSRHIPYLAEGQQIAEQF]₈K₄K₂K-COOH (MDL022)Synthesis of Multiple Antigenic Peptide MDL029

In order to work successfully, the serotyping assay requires thatpeptides are synthesized on a special resin support, bearing themultiple antigen peptide core (K₄K₂K) as developed by Tam (Tam (1988)Proc. Natl. Acad. Sci. USA. 85:5409:5413). All peptides were synthesizedon an Applied Biosystems model 432A Synergy peptide synthesizer runningFASTmoC™ chemistry. Peptide synthesis was achieved using the standardrun program without modification. All reagents used were supplied byApplied Biosystems Limited (Kelvin Close, Birchwood Science Park,Warrington, UK). The MAP resin was hepta-lysyl (K₄K₂K) core on apolyoxyethylene/polystyrene co-polymer with an HMP linker andbeta-alanine internal reference amino acid. The N-a-amino group of allamino acids was protected by the 9-fluorenylmethoxycarbonyl (Fmoc)group. Amino acids with reactive side groups were protected as follows:

Amino Acid Code Protecting Group glutamine Q tert. Butyl ester (OtBu)cysteine C trityl (Trt) serine S tert. Butyl (tBu) threonine T tert.Butyl (tBu) tyrosine Y tert. Butyl (tBu) aspartic acid D tert. Butylester (OtBu) arginine R 2,2,5,7,8-Pentamethylchroman-6-sulphonyl (Pmc)

The progress of the synthesis was monitored by measuring theconductivity of the coupling and deprotection mixtures. There are noabnormalities in the conductivity trace and the synthesis was consideredsuccessful.

Following the synthesis, the fully protected peptide resin wastransferred to a conical 50 mL capacity polypropylene tube and treatedwith thioanisole (0.15 mL), ethandithiol (0.15 mL) and trifluoroaceticacid (TFA; 2.7 mL). The mixture was stirred for three hours at roomtemperature, then filtered through glass wool in a Pasteur pipette, thefiltrate dropping into 20 mL tert. butylmethylether (TMBE) contained ina glass screw-capped bottle, whereupon the peptide precipitated. Thetube was centrifuged and the liquor aspirated, leaving the peptidepellet in the tube. Peptide was washed with three further 20 mL aliquotsof TMBE, using a spatula to dis-aggregate the pellet and centrifugationto recover the peptide. The peptide was finally dried at roomtemperature in vacuo.

To analyze the purity of the peptide, a small sample was dissolved inpurified water and submitted to analysis by reverse phasehigh-performance liquid chromatography (HPLC). The reverse phase columnwas 250×4.6 mm containing a C₄ matrix. Peptide was eluted with agradient of 3.5% acetonitrile to 70% acetonitrile over 20 minutes at aflow rate of 1.5 mL min⁻¹. The eluant was monitored at 214 nm to detectpeptide.

EXAMPLE 4 Assay for the Determination of HCV Serotypes 1-6

We have developed an assay based on selective competition, which iscapable of distinguishing the serotype of HCV to which antibodiespresent in a biological sample have been produced. Typically the samplewill be serum or plasma from a human with confirmed hepatitis Cinfection.

The assay relies on the selectivity of 17 different synthetic peptidescovering variable sequences within the NS4 protein of hepatitis C virustypes 1, 2, 3, 4, 5 and 6. Whilst there is a degree of cross-reactivitybetween antisera raised to NS4 of an HCV serotype and the homologousregion of other serotypes, this can be blocked without completelyremoving the true reactivity. In view of this, we have developed anassay format involving coating wells of a microtiter plate with an equalamount of all 17 peptides. Samples are tested in eight duplicate wellswhich each receive a different mixture of blocking peptides. Only onetesting well and the un-completed control well should contain colorationat the end of the assay protocol, thereby identifying the serotype ofthe infecting hepatitis C virus.

Plates for serotyping assays are prepared by coating approximatelyequimolar amounts of each of the peptides onto polystyrene microwellplates. Peptides are dissolved in purified water and a mixture is madeat the following concentrations:

25 ng/ml 50 ng/ml MDL031 MDL039 MDL033 MDL041 MDL036 MDL040 MDL035MDL042 MDL037Q MDL044 MDL038Q MDL034 MDL028 MDL024 MDL029 MDL025 MDL022

Although strictly speaking each microwell need only contain peptide ofthe type which it is intended to detect, it is more convenient to coateach microwell with all six antigen types. Peptides were allowed to bindto the plates by adding 100 ul of the peptide mixture into each well ofthe plate and incubating at +4 degree C. overnight. To providesufficient differentiation between the various serotypes, it is thennecessary to add competing heterologous peptides to the sample beingtested. Competing solutions are made up in assay sample diluent to givea 100-fold excess of competing peptide, relative to the coatingconcentration. The different competing solutions are classifiedaccording to the serotype for which they are blocking, i.e. CompetingSolution 1 contains peptides for types 2-5. Competing solutions are madeto have the excess required in 10 ul. The competing solutions are asfollows:

Competing solution Peptide Concentration 1 MDL039 50 ug/ml each MDL041MDL040 MDL042 MDL044 MDL034 MDL028 MDL024 MDL029 MDL025 MDSL022 2 MDL03125 ug/ml MDL033 25 ug/ml MDL036 25 ug/ml MDL035 25 ug/ml MDL037Q 25ug/ml MDL038Q 25 ug/ml MDL042 50 ug/ml MDL044 50 ug/ml MDL034 50 ug/mlMDL028 50 ug/ml MDL024 50 ug/ml MDL029 50 ug/ml MDL025 50 ug/ml MDL02250 ug/ml 3 MDL031 25 ug/ml MDL033 25 ug/ml MDL036 25 ug/ml MDL035 25ug/ml MDL037Q 25 ug/ml MDL038Q 25 ug/ml MDL039 50 ug/ml MDL040 50 ug/mlMDL041 50 ug/ml MDL034 50 ug/ml MDL028 50 ug/ml MDL024 50 ug/ml MDL02950 ug/ml MDL025 50 ug/ml MDL022 50 ug/ml 4 MDL031 25 ug/ml MDL033 25ug/ml MDL036 25 ug/ml MDL035 25 ug/ml MDL037Q 25 ug/ml MDL038Q 25 ug/mlMDL039 50 ug/ml MDL040 50 ug/ml MDL041 50 ug/ml MDL042 50 ug/ml MDL04450 ug/ml MDL024 50 ug/ml MDL029 50 ug/ml MDL025 50 ug/ml MDL022 50 ug/ml5 MDL031 25 ug/ml MDL033 25 ug/ml MDL036 25 ug/ml MDL035 25 ug/mlMDL037Q 25 ug/ml MDL038Q 25 ug/ml MDL039 50 ug/ml MDL040 50 ug/ml MDL04150 ug/ml MDL034 50 ug/ml MDL028 50 ug/ml MDL042 50 ug/ml MDL044 50 ug/mlMDL025 50 ug/ml MDL022 50 ug/ml 6 MDL031 25 ug/ml MDL033 25 ug/ml MDL03625 ug/ml MDL035 25 ug/ml MDL037Q 25 ug/ml MDL038Q 25 ug/ml MDL039 50ug/ml MDL040 50 ug/ml MDL041 50 ug/ml MDL034 50 ug/ml MDL028 50 ug/mlMDL024 50 ug/ml MDL029 50 ug/ml MDL042 50 ug/ml MDL044 50 ug/ml

The protocol for using the serotyping assay is as follows:

-   -   1) Add 180 ul sample of diluent to each well.    -   2) Add 10 ul blocking peptides to relevant wells.    -   3) Add 10 ul sample to each of six wells.    -   4) Mix plate and incubate at 37 degree C. for 1 hour.    -   5) Wash wells three times.    -   6) Add 100 ul conjugate to each well.    -   7) Incubate at 37 degree C. for 1 hour.    -   8) Wash wells three times.    -   9) Add 100 ul TMB solution (including        3,31,5,5′-tetramethylbenzidine, hydrogen peroxide, buffers etc).    -   10) Incubate at 37 degree C. for 30 minutes.    -   11) Stop reaction with sulphuric acid; and    -   12) Read optical density at 450 nm/690 nm.

Samples known to contain anti-HCV antibodies are tested at a dilution of1/20 with 100 fold excess of competing peptides, in a total volume of200 ul as described above. Following incubation, sample is removed andthe wells are washed. An anti-human immunoglobulin G conjugated tohorseradish peroxidase is added, and binds to any captured anti-HCVantibodies. Bound antibody is then visualised by removing the enzymeconjugate and adding a substrate and chromogen, which, in the presenceof enzyme, converts from a colorless to a colored solution. Theintensity of the color can be measured and is directly proportional tothe amount of enzyme present. Results on certain samples are given inTable 1.

TABLE 1 NO ALL SAMPLE BLOCK BLOCK TYPE 1 TYPE 2 TYPE 3 TYPE 4 TYPE 5TYPE 6 RESULT Control 1 0.515 0.049 0.530 0.033 0.038 0.038 0.031 0.0291 Control 2 1.821 0.010 0.028 1.916 0.004 −0.007  0.040 0.005 2 Control3 2.035 0.066 0.048 0.046 1.933 0.048 0.067 0.044 3 Egypt 3 OVER 0.3720.346 0.478 0.322 OVER 0.256 0.430 4 C1016116 OVER 0.456 0.635 0.4800.533 0.533 OVER 0.617 5 Control 6 0.082 0.004 0.007 −0.003  −0.004 −0.012  −0.011  0.067 6 PD372 OVER 0.076 1.497 0.081 0.072 0.040 0.0570.075 1 PD513 OVER 0.548 0.467 OVER 0.553 0.470 0.525 0.582 2 AD558 OVER0.031 0.064 0.044 OVER 0.035 0.054 0.048 3 Egypt 37 OVER 0.034 0.0640.039 0.045 1.473 0.047 0.037 4 C1015921 0.173 0.010 0.017 0.008 0.0110.005 0.182 0.012 5 Positive results are shown in bold text.

EXAMPLE 5 Assay for the Determination of HCV Serotypes 4-6

An alternative to the assay format in Example 4 has been developed. Thisassay is more limited in scope using only the peptides for types 4, 5and 6. For some samples, the more comprehensive assay may give erroneousresults, due to greater cross-reactivity with type 1 peptides. Theprotocol for performing the more restricted assay is identical to thatgiven in Example 4.

Plates for the HCV types 4-6 assay are prepared as described in Example4 with the only difference being the reduced number of peptides used.Peptides are dissolved in purified water and a mixture is made at thefollowing concentration:

MDL034 50 ng/ml MDL028 MDL024 MDL029 MDL025 MDL022Peptides are allowed to bind to the plates by adding 100 ul of thepeptide mixture into each well of the plate and incubating at +4 degreeC. overnight.

To provide sufficient differentiation between the various serotypes, itis then necessary to add competing heterologous peptides with the samplebeing tested. Competing solutions are made up in assay sample diluent togive a 100-fold excess of competing peptide, relative to the coatingconcentration.

The different competing solutions are classified according to theserotype for which they are blocking, i.e. Competing Solution 4 containspeptides for types 5 and 6. Competing solutions are made to have theexcess required in 10 ul. The competing solutions are as follows:

Competing Solution Peptide Concentration 4 MDL024 50 ug/ml each MDL029MDL025 MDL022 5 MDL034 50 ug/ml each MDL028 MDL025 MDL022 6 MDL034 50ug/ml each MDL028 MDL024 MDL029

Samples known to contain anti-HCV antibodies are tested at a dilution of1/20 with 100 fold excess of competing peptides, in a total volume of200 ul, as described in Example 4. Following incubation, sample isremoved and the wells are washed. An anti-human immunoglobulin Gconjugated to horseradish peroxidase is added, and binds to any capturedanti-HCV antibodies. Bound antibody is then visualised by removing theenzyme conjugate and adding a substrate and chromogen, which, in thepresence of enzyme, converts from a colorless to a colored solution. Theintensity of the color can be measured and is directly proportional tothe amount of enzyme present. Results on certain samples are given inTable 2.

TABLE 2 SAMPLE NO BLOCK ALL BLOCK TYPE 4 TYPE 5 TYPE 6 EGYPT 1 OVER0.109 1.669 0.184 0.048 EGYPT 2 0.525 0.010 0.514 0.001 −0.006 EGYPT 3OVER 0.269 OVER 0.329 0.201 EGYPT 4 0.266 −0.001  0.292 0.006 −0.002EGYPT 5 1.677 0.090 1.274 0.094 0.086 EGYPT 6 OVER 0.208 0.317 0.0410.022 EGYPT 7 OVER 0.113 OVER 0.143 0.204 EGYPT 8 OVER 0.085 0.891 0.1050.098 EGYPT 9 0.051 0.009 0.055 0.014 0.022 EGYPT 10 OVER 0.021 2.0230.043 0.073 EGYPT 11 0.077 0.024 0.095 0.032 0.048 C1016116 OVER 0.6470.778 OVER 0.513 C1015921 0.196 0.014 0.009 0.222 0.009 C1132558 0.0150.007 0.008 0.015 0.007 K904836 OVER 0.073 0.101 OVER 0.071 Control 60.433 0.029 0.013 0.015 0.314 HK T3950 0.428 0.034 0.017 0.027 0.366 HKT3943 OVER 0.348 0.424 0.342 1.105 Positive results are shown in boldtext. NB - The preponderance of Type 4 results reflects the relativeincidence and availability or samples for types 4, 5, & 6.

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1. An isolated polynucleotide sequence comprising a nucleotide sequenceselected from the group consisting of: a) the nucleotide sequence of SEQID NO:49; and b) a nucleotide sequence encoding the amino acid sequenceof SEQ ID NO:50.